Coregulation of Dimorphism and Symbiosis by Cyclic AMP Signaling in the Lichenized Fungus Umbilicaria Muhlenbergii

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Coregulation of dimorphism and symbiosis by cyclic AMP signaling in the lichenized fungus Umbilicaria muhlenbergii

Yanyan Wanga,b, Xinli Weia, Zhuyun Bianb, Jiangchun Weia, and Jin-Rong Xub,1

aState Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, China; and bDepartment of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907

Edited by Nicholas J. Talbot, The Sainsbury Laboratory, Norwich, United Kingdom, and accepted by Editorial Board Member Sheng Yang He August 10, 2020 (received for review March 18, 2020) Umbilicaria muhlenbergii is the only known dimorphic lichenized (8). In fact, ∼46% of ascomycetes are lichen mycobionts (9), and fungus that grows in the hyphal form in lichen thalli but as yeast a large majority of them belong to class Lecanoromycetes (8). cells in axenic cultures. However, the regulation of yeast-to-hypha For most lichenized fungi, their free-living forms are rarely ob- transition and its relationship to the establishment of symbiosis served in nature (10), although they may be cultured axenically are not clear. In this study, we show that nutrient limitation and under laboratory conditions. In general, the isolated mycobionts hyperosmotic stress trigger the dimorphic change in U. muhlen- grow very slowly in culture and may require weeks of incubation bergii. Contact with algal cells of its photobiont Trebouxia jamesii to form sizable colonies, which are typically compact and pig- induced pseudohyphal growth. Treatments with the cAMP diphos- mented (11). Among all lichenized ascomycetes (>280 genera) phoesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) induced that have been isolated, only a few species are known to produce pseudohyphal/hyphal growth and resulted in the differentiation conidia and ascospores in culture (12–14). of heavily melanized, lichen cortex-like structures in culture, indi- Resynthesis of a lichen thallus is a complex process that begins cating the role of cAMP signaling in regulating dimorphism. To with the physical contact between the mycobiont and photobiont confirm this observation, we identified and characterized two cells. The two symbiotic partners then grow together to form an Gα subunits UmGPA2 and UmGPA3. Whereas deletion of UmGPA2

undifferentiated mass, which further develops into a stratified MICROBIOLOGY had only a minor effect on pseudohyphal growth, the ΔUmgpa3 thallus after a transitional stage (6, 15). The formation of a mature mutant was defective in yeast-to-pseudohypha transition induced lichen thallus is difficult under laboratory conditions, likely due to by hyperosmotic stress or T. jamesii cells. IBMX treatment sup- the requirement of unknown specific environmental or biological pressed the defect of ΔUmgpa3 in pseudohyphal growth. Trans- factors in nature. However, the initial interaction between the G45V Q208L formants expressing the UmGPA3 or UmGPA3 dominant mycobiont and photobiont and structural development have been active allele were enhanced in the yeast-to-pseudohypha transition reported in several species (16–19). Before the physical contact and developed pseudohyphae under conditions noninducible to the stage, the mycobiont exhibits selectivity and compatibility toward wild type. Interestingly, T. jamesii cells in close contact with pseu- – G45V Q208L different phototrophs (20 22). At the interaction stage, fungal dohyphae of UmGPA3 and UmGPA3 transformants often hyphae often increase the production of short lateral branches to collapsed and died after coincubation for over 72 h, indicating that envelop compatible algal cells (23). Some mycobionts, such as improperly regulated pseudohyphal growth due to dominant active Cladonia, Lecanora,andXanthoria, form haustoria or haustorium-like mutations may disrupt the initial establishment of symbiotic interaction between the photobiont and mycobiont. Taken together, Significance these results show that the cAMP-PKA pathway plays a critical role in regulating dimorphism and symbiosis in U. muhlenbergii. Umbilicaria muhlenbergii istheonlylichenizedspeciesknowntobe cAMP signaling | lichen-forming fungi | dimorphic transition | dimorphic in culture. This study showed that yeast-to-pseudohypha yeast-to-hypha transition | fungal–algal association transition in U. muhlenbergii is associated with symbiosis and regulated by the cAMP-PKA (protein kinase A) pathway. Treatments with a cAMP diphosphoesterase inhibitor induced pseudohyphal/ ichens are symbiotic associations between a fungus (myco- hyphal growth and differentiation of lichen cortex-like tissues. Two Lbiont) and a photosynthetic partner (photobiont), usually Gα subunits were functionally characterized to show the regulation green algae or cyanobacteria. Apart from the two major symbi- of dimorphism by UmGPA3. This is a report on identifying genes onts, other organisms, including bacteria, fungi, and algae, may be important for development and symbiosis in lichenized fungi. This is present and have functions in lichens as a stable symbiotic com- also a report of generating targeted gene deletion mutants in U. munity (1–3). Lichens can thrive in harsh environments and play ∼ muhlenbergii and Lecanoromycetes in general. Because of its rela- important roles in the ecosystem by covering 8% of the terres- tively fast growth rate and amenability to molecular manipulations, trial surface (4, 5). Based on the morphology of their thalli, lichens U. muhlenbergii is uniquely suited for studying fungal–algal inter- can be categorized into different growth types, such as fruticose, actions and initial symbiotic interactions. foliose, and crustose lichens. Typically, lichens have a cortex or outer layer that consists of highly differentiated, densely aggre- Author contributions: J.W. and J.-R.X. designed research; Y.W., X.W., and Z.B. performed gated fungal hyphae or symbiotic complex in which individual research; Y.W., X.W., and Z.B. analyzed data; and Y.W., J.W., and J.-R.X. wrote the paper. hyphae are no longer distinguishable (6, 7). Beneath the upper The authors declare no competing interest. cortex is a layer of photobiont cells that may be arranged in dis- This article is a PNAS Direct Submission. N.J.T. is a guest editor invited by the order or regularly with interwoven hyphae. For most lichens, the Editorial Board. lower surface or cortex may form special structures for tight ad- Published under the PNAS license. herence to the substrate. 1To whom correspondence may be addressed. Email: [email protected]. Although lichenization occurs in species belonging to poly- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ phyletic groups in different phyla, indicating multiple origins of doi:10.1073/pnas.2005109117/-/DCSupplemental. lichenized fungi, the majority (99%) of them are ascomycetes

www.pnas.org/cgi/doi/10.1073/pnas.2005109117 PNAS Latest Articles | 1of12 Downloaded by guest on September 27, 2021 structures within photobiont cells (21, 24). However, due to their of the top layers, U. muhlenbergii strain JL3 was isolated from slow growth rate, lichenized fungi are not ready amenable to the medullary layer of lichen thalli collected at Tulaopoding molecular genetic studies. To date, no genes responsible for the Mountain, China. As previously reported (25), strain JL3 grew by establishment of symbiosis have been functionally characterized budding as yeast cells (3.0 to 4.5 × 5.0 to 6.5 μm) in potato at the molecular level. dextrose agar cultures (SI Appendix, Fig. S2). After incubation at Unlike all other cultivated lichen-forming ascomycetes that 25 °C for 14 d, strain JL3 formed whitish colonies with a smooth grow only as hyphae, the isolated mycobiont of the lichen, margin. BLASTn searches and phylogenetic analysis with the Umbilicaria muhlenbergii, grows by budding as yeast cells in axenic rRNA (ribosomal RNA)-ITS (internal transcribed spacer) se- cultures (25). U. muhlenbergii is a foliose lichen that usually grows quence (ITS1 + 5.8S rRNA + ITS2) amplified with primers ITS4 on bare stones at high altitudes. Its photobiont partner is mainly and ITS5 (33) confirmed that it is a U. muhlenbergii isolate (SI Trebouxia jamesii, a green algal species (26). The unicellular yeast Appendix,Fig.S3). The rRNA-ITS sequence of U. muhlenbergii cells of U. muhlenbergii mycobiont grow by budding and form strain JL3 is identical to that of the published strain (25, 34). typical yeast-like colonies (25). Although U. muhlenbergii lives in We also isolated the green algal photobiont T. jamesii from the the yeast form in culture, it exists in the hyphal or pseudohyphal same specimens and verified its identity by sequencing the form in the lichen thallus, indicating that this lichenized fungus rRNA-ITS region (SI Appendix, Fig. S3). Algal cells of T. jamesii must undergo the yeast-to-hypha transition during the establish- are spherical and 10 to 15 μm in diameter. Another green algal ment of symbiosis. U. muhlenbergii, like many other species in the species with a different colony morphology and bigger algal cells genus Umbilicaria, can grow in extreme environmental conditions than T. jamesii was repeatedly isolated from the same lichen and often produces heavily melanized thalli. Ascospores formed in thallus (SI Appendix, Fig. S2), which was not observed in the apothecia (sexual fruiting bodies) are its preferred propagules for earlier report (27). Analysis of the rRNA-ITS sequences showed dispersal instead of the vegetative reproducing mixtures of that it is an Elliptochloris species closely related to Elliptochloris mycobiont and photobiont cells (27). Other than ascospores, the subsphaerica (SI Appendix, Fig. S3). Elliptochloris species have U. muhlenbergii lichen thalli also can produce spermatia on sper- been reported as endolichenic algae present in various lichens (3, matiophores in spermogonia (asexual fruiting bodies). Unicellular 35). In comparison with T. jamesii, this Elliptochloris isolate had a spermatia are rod like (4.8 × 2.4 μm) and hyaline (28). Although faster growth rate and required higher light intensity for growth. some Umbilicaria species produce thalloconidia that are often We also isolated the mycobiont from lichen thalli of U. darkly pigmented and multicellular (multiseptate) directly from muhlenbergii collected from Smoky Mountain, NC. Similar to the scleroplectenchymatous tissues or protruding rhizinomorphs of Chinese isolates, the US isolate (US1) also grew as yeast cells on the lower cortex of lichen thalli, U. muhlenbergii is one of the regular PDA media and is in the same clade with known U. Umbilicaria species that does not form thalloconidia (29–32). Even muhlenbergii isolates based on phylogenetic analysis (SI Appen- the formation of spermatia and spermogonia observed in the dix, Fig. S3). symbiotic state (lichen thalli) has not been observed in culture, and their biological functions in the life cycle of U. muhlenbergii Nutrient Limitation Induces Pseudohyphal Growth in U. muhlenbergii. remain to be characterized. U. muhlenbergii is a widely distributed Because of its slow growth rate, we normally examined yeast cells fungus in northern Asia and North America (32). However, to and colony morphology of U. muhlenbergii after incubation for date, only one U. muhlenbergii strain isolated from China has been 14 d (Fig. 1C). We noticed that colony morphology changed in observed to undergo dimorphic transition in axenic cultures (25), old cultures. In 1-mo-old cultures, colonies of U. muhlenbergii and the underlying mechanism regulating dimorphism is not clear. became brown and wrinkled. Microscopical examination showed Whereas dimorphism is known to be related to pathogenesis in that some cells had undergone the yeast-to-hypha transition, and some fungal pathogens, U. muhlenbergii is the only lichenized pseudohyphae were visible. fungus known to be dimorphic under laboratory conditions and We hypothesized that pseudohyphal growth was induced by thus, is uniquely suitable for studying the relationship between and nutrient starvation in old cultures. To test this hypothesis, we coregulation of dimorphism and symbiosis. In this study, we show cultured U. muhlenbergii on dilute PDA medium. On 0.2× PDA that nutritional and environmental stresses as well as algal cells of plates, colonies became brown and had an uneven surface after its photobiont induced the yeast-to-pseudohypha transition in U. incubation for 14 d (Fig. 1C). A large number of yeast cells were muhlenbergii. Treatment with cAMP and IBMX (3-isobutyl-1- found to have switched to pseudohyphal growth. Daughter cells methylxanthine; an inhibitor of cAMP diphosphoesterase) also were not separated from mother cells and elongated, forming stimulated pseudohyphal growth and the differentiation of lichen pseudohyphae that had budding tip cells (Fig. 1D). cortex-like structures in culture, indicating a regulatory role of To further test that nutrient limitation induces pseudohyphal cAMP signaling (SI Appendix,Fig.S1). Whereas mutants deleted growth, we cultured U. muhlenbergii on the inorganic ion me- of the UmGPA3 Gα gene that functions upstream from the dium BBM (Bold’s Basal Medium) (36) used for algal cultures. cAMP-PKA pathway were defective in the yeast-to-pseudohypha Due to the lack of a carbon source, fungal growth on BBM was transition, expressing dominant active alleles of UmGPA3 en- limited, but pseudohyphal development was observed as early as hanced pseudohyphal growth and had a detrimental effect on T. 5 d (Fig. 1E). After incubation on BBM for 1 mo, pseudohyphae jamesii algal cells in close contact with fungal pseudohyphae. became extremely long and produced unicellular, rod-shaped These results indicate that the cAMP-PKA pathway plays a critical conidia (1.5 to 2.0 × 4.0 to 5.0 μm) that were smaller than typ- role in regulating dimorphism and the initial symbiotic interaction ical yeast cells (Fig. 1 E and H). Conidia were produced and with its photobiont cells in the Lecanonormycete U. muhlenbergii. aggregated at the tip or branching site of long pseudohyphae, which was not observed in old cultures on 0.2× PDA. Results Isolation of Mycobiont and Photobiont. The lichen U. muhlenbergii Hyperosmotic Stress Also Induces Pseudohyphal Growth. In aged normally has a circular thallus with umbilici at the center of the cultures, U. muhlenbergii may develop pseudohyphae in response to lower surface for substrate attachment and often forms apoth- hyperosmotic stress caused by the desiccation of PDA media. To ecia on mature thalli (Fig. 1A). Beneath the highly differentiated test this hypothesis, we first assayed growth on PDA supplemented upper cortex is the algal layer and medullary layer (Fig. 1A). with 1 M sorbitol, 1 M NaCl, or 20% sucrose. After incubation for Close contacts between branching hyphae of the mycobiont and 10 d, pseudohyphal growth was observed on PDA with 1 M sorbitol, algal cells of the photobiont were observed at the interface of but the overall fungal growth was limited, and many cells appeared these two layers (Fig. 1B). After surface sterilization and removal to be dead or devoid of cytoplasm (SI Appendix,Fig.S4). Similar

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Fig. 1. Lichen thallus, colony morphology, and dimorphism of U. muhlenbergii.(A) The lichen thallus and its cross-section to show the upper cortex (UC), photobiont layer (P), medulla layer (M), and lower cortex (LC). Apothecia (sexual fruiting bodies) are formed on the upper surface. The undersurface has the umbilicus (a central holdfast) for attaching to rocks. (B) The cross-section of a lichen thallus examined by scanning electron microscopy. Hyphae of the mycobiont (MY) wrap around algal cells of the photobiont (PH). (C) Colonies of U. muhlenbergii on regular and diluted potato dextrose agar (0.2× PDA). (D) Yeast cells grown on PDA and pseudohyphae grown on 0.2× PDA were examined by DIC (differential interference contrast) and scanning electron microscopy (SEM). (E) Colonies and pseudohyphae of U. muhlenbergii on BBM plates. Pseudohyphae and conidia were examined after staining with CFW. (F) Colonies, close-up view of colony margins, and pseudohyphae of U. muhlenbergii cultured on PDA with 0.5 or 0.8 M sorbitol. Fuzzy surfaces and margins are related to pseudohyphal growth. (G) Pseudohyphae induced by 0.5 M sorbitol were examined by DIC and scanning electron microscopy (SEM). (H) Size and morphology of yeast cells, pseudohyphae, and conidia of U. muhlenbergii. The incubation time was 14 d for regular PDA, 0.2× PDA, or PDA with 0.5/0.8 M sorbitol and 1 mo for BBM.

Wang et al. PNAS Latest Articles | 3of12 Downloaded by guest on September 27, 2021 results were obtained when U. muhlenbergii was cultured on PDA growth of U. muhlenbergii was induced by algal cells of T. jamesii with 1 M NaCl or 20% sucrose. These results indicated that the during their early interactions, but they failed to establish a osmotic stress caused by 1 M sorbitol, 1 M NaCl, or 20% sucrose stable symbiotic relationship as in the lichen thallus under this induces pseudohyphal growth but is harmful to U. muhlenbergii. laboratory condition. We then assayed for pseudohyphal growth on PDA with 0.5 or 0.8 M sorbitol. After incubation for 14 d, the overall growth of U. Algal Cells of Elliptochloris Fail to Induce Pseudohyphal Growth in U. muhlenbergii was much more robust on medium with 0.5 M muhlenbergii. To determine whether the Elliptochloris strain iso- sorbitol than on medium with 0.8 M sorbitol, confirming its lated in this study induces dimorphic transition in U. muhlenbergii, sensitivity to hyperosmotic stress. Colonies with fuzzy margins we also coincubated mixtures of these two organisms as described and an uneven surface were observed in both conditions, al- above. In their cocultures, pseudohyphal growth and changes in though 0.8 M sorbitol appeared to be more effective in inducing yeast cell morphology were not observed in U. muhlenbergii cells in pseudohyphal growth (Fig. 1F). Pseudohyphae induced by 0.5 or close contact with Elliptochloris cells (SI Appendix,Fig.S6), and 0.8 M sorbitol were longer and more highly branched than they could be easily separately by a gentle touch or washing with pseudohyphae formed on old PDA cultures (Fig. 1F). In com- sterile water. Algal cells of this Elliptochloris isolate grew faster parison with normal yeast cells, cellular compartments in pseu- that T. jamesii but failed to induce the yeast-to-hypha morpho- dohyphae were narrower but longer, and normally, only the tip logical transition in U. muhlenbergii, indicating a specific recog- cell still grew by budding. Lateral growth from some compart- nition between the mycobiont and T. jamesii cells. ments resulted in the branching of pseudohyphae (Fig. 1G). When stained with 4′,6-diamidino-2-phenylindole and Calco- Exogenous cAMP and IBMX Stimulate the Development of Pseudohyphae. fluor white (CFW) to visualize the nucleus and cell wall, the Because of the conserved role of the cAMP-PKA pathway in in- unicellular yeast cells of U. muhlenbergii had a single nucleus. tracellular signaling and nutrient sensing in fungi, we assayed the Pseudohyphae induced by nutrient starvation or osmotic stress effect of exogenous cAMP on U. muhlenbergii. The addition of also had one nucleus in each compartment (SI Appendix, Fig. 10 mM cAMP affected colony morphology and stimulated the S5). These results indicated that, although the compartments in elongation of yeast cells that was visible as early as 48 or 72 h on pseudohyphae were longer than normal yeast cells of U. muh- PDA plates (Fig. 3A). After incubation for 14 d, colonies of U. lenbergii, they were still uninucleate. Pseudohyphae appeared to muhlenbergii often had a bumpy or uneven surface and some areas grow by budding at the tip, and each septum was associated with with increased pigmentation due to pseudohyphal growth (Fig. 3A). a constriction at the budding site of the growing tip cell. We then tested the effect of IBMX, an inhibitor of cAMP diphosphoesterase, on U. muhlenbergii. Discs of Whatman filter Oxidative and Cell Wall Stresses Fail to Induce Pseudohyphal Growth. paper containing 25 nmol of IBMX (dissolved in dimethyl sulf- We tested the impact of oxidative and cell wall stress on pseu- oxide) were placed on one side of a streak of U. muhlenbergii cells dohyphal growth in U. muhlenbergii. This fungus appears to be across the surface of 10-mL PDA plates. As IBMX diffused in the hypersensitive to all these stresses (SI Appendix, Fig. S4). In the medium, it formed a descending concentration gradient across the presence of 1 mM H2O2, growth was limited, and most of the U. muhlenbergii colony (Fig. 3B). Stimulation of colony pigmen- cells were dead (empty) after incubation for 10 d. On PDA tation (Fig. 3B) and pseudohyphal growth (Fig. 3C) were observed containing 1.5 mM Congo Red, U. muhlenbergii grew slightly at 7 d post–3-isobutyl-1-methylxanthine treatment (dpt). At 14 better, but many dead (empty) yeast cells also were observed. dpt, the surface of U. muhlenbergii colony near the filter paper Unlike hyperosmotic stress, stimulation of pseudohyphal growth (0.5 cm away from the filter paper) became darkly pigmented with by oxidative and cell wall stresses was not observed. a bumpy and hairy appearance. When the dark-pigmented clumps (tissue-like structures) were crushed, aggregates of pseudohyphae Pseudohyphal Growth Is Induced by T. jamesii Cells. Because U. or hyphae and heavily melanized spherical cells were observed muhlenbergii is in the hyphal form in the lichen thallus, we tested (Fig. 3 B and C). These spherical cells are likely related to dif- the effect of algal cells on the yeast–hypha dimorphic transition. ferentiated cells or compartments of pseudohyphae. When cells of T. jamesii were mixed with U. muhlenbergii cells and After incubation for 1 mo, IBMX induced more significant coincubated on PDA, attachment of yeast cells to algal cells and changes in U. muhlenbergii, particularly in areas near the filter induction of pseudohyphal growth were observed (Fig. 2A). Pseu- paper. The bumpy colonies were heavily melanized and covered dohyphae were visible around algal cells after cocultivation for 72 h with protruding hyphae or long pseudohyphae (Fig. 3D). Micro- (Fig. 2A), and the resulting fungal–algal cell aggregates could not scopic examination showed that the highly differentiated, hard- be easily separated by a gentle touch. When yeast cells of U. ened tissues consisted of a dense epidermal layer with heavily muhlenbergii were resuspended to 5 × 103/mL in the supernatant of melanized pseudohyphae or yeast cells, in which individual com- BBM cultures of T. jamesii after removing algal cells by centrifu- partments or cells were often no longer distinguishable. Further- gation and cultured on PDA plates, pseudohyphal growth as shown more, hyphal filaments without obvious constrictions also were in Fig. 2A was not observed after incubation for 72 h, indicating the often observed in these highly differentiated tissues. Pseudohyphal importance of physical contact between fungal and algal cells. and hyphal growth on the surface of IBMX-treated colonies also After coincubation for 14 d, long pseudohyphae with growing was observed by scanning electron microscopy (Fig. 3D). There- tip cells were formed (Fig. 2B). Some of these pseudohyphae had fore, IBMX was more effective in inducing pseudohyphal and the tip cell growing away from algae, although their basal ends hyphal growth than cAMP. The development of an epidermal aggregated around T. jamesii cells (Fig. 2B). The close contact layer of highly differentiated and melanized hyphal compartments between fungal and algal cells was observed after staining with or yeast cells on the clumps of IBMX-treated colonies was, similar CFW (Fig. 2C). Interestingly, compartments of pseudohyphae to the cellular differentiation of U. muhlenbergii, induced by the tended to vary significantly in size and morphology in the photobiont in the lichen thalli. hyphal–algal aggregates but became more uniform when pseudohyphae were not in close contact with algae (Fig. 2 B and C). The UmGPA3 Gene Regulates Pseudohyphal Growth. The Gα subunits After incubation for 14 d, the surface of fungal–algal cocultures of the trimeric G proteins that function upstream from the cAMP- had a hairy appearance that is related to pseudohyphal growth of PKA pathway have been characterized in different fungi for roles in U. muhlenbergii (Fig. 2D). Enhanced pigmentation and pockets morphogenesis and yeast–hypha dimorphism (37, 38). A BLASTP of green algal cells also were observed in some parts of the co- search of the U. muhlenbergii genome (34) with three Magnaporthe cultures (Fig. 2D). These results indicated that pseudohyphal oryzae Gα subunits identified three orthologs on contigs 70, 79, and

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Fig. 2. Pseudohyphal growth of U. muhlenbergii induced by its photobiont T. jamesii.(A) Time course assays of the initial interaction of U. muhlenbergii yeast cells with its photobiont T. jamesii.(B) Pseudohyphae of U. muhlenbergii after coculturing with T. jamesii for 2 wk. (C) Pseudohyphae induced by algal cells were stained with CFW and examined by epifluorescence microscopy. (D) A mixed culture of U. muhlenbergii and T. jamesii cells after incubation for 14 d on PDA.

232, respectively. They were named as UmGPA1 (GenBank acces- cells were pseudohyphae (Fig. 4B). However, the ΔUmgpa3 sion no. MT50998), UmGPA2 (GenBank accession no. MT509979), mutant still grew by budding as yeast cells (Fig. 4B), indicating and UmGPA3 (GenBank accession no. MT509978) in this study. that deletion of UmGPA3 affected the yeast-to-hypha transition Sequence alignment and phylogenetic analysis showed that on nutrient-limited media. UmGpa3 is homologous to Ustilago maydis Gpa3 and Candida We also assayed the defect of ΔUmgpa3 mutant in dimorphic albicans Gpa2 that are involved in regulating dimorphism and transition induced by hyperosmotic stress. On PDA with 0.5 M pathogenesis. UmGpa2 is in the same clade with U. maydis Gpa2 sorbitol, the wild-type strain formed wrinkly colonies with fuzzy and C. albicans Gpa1 (SI Appendix,Fig.S7). margins, but ΔUmgpa3 colonies had a smooth appearance Based on the functions of orthologs characterized in other (Fig. 4C). Microscopic examination of cells from colony margins fungi, such as U. maydis Gpa3 and C. albicans Gpa2 (37, 39), we showed that the wild type had long and branching pseudohyphae hypothesized that UmGPA3 may play a more important role in induced by 0.5 M sorbitol. Under the same conditions, no pseu- dimorphism than the other Gα subunits in U. muhlenbergii.To dohyphal growth was observed in the ΔUmgpa3 mutant (Fig. 4C determine its function, the Umgpa3 gene knockout mutant was and SI Appendix,Fig.S10). generated by homologous recombination using the split-marker To further verify that UmGPA3 functions upstream from the approach (SI Appendix,Fig.S8) (40). After screening hygromycin cAMP-PKA pathway, 2.5 mM IBMX was added to PDA cultures -resistant transformants generated in five independent transfor- of the ΔUmgpa3 mutant. Pseudohyphal growth induced by mations, three independent Umgpa3 deletion mutants (gpa3-1, IBMX was visible in the ΔUmgpa3 mutant after incubation for gpa3-3, and gpa3-4) were identified and confirmed by PCR with 4 d (Fig. 4E). After incubation for 14 d, the highly differentiated anchor primers. All three Umgpa3 deletion mutants had the same and melanized pseudohyphae were observed in the hairy clumps phenotype, although only data of mutant strain gpa3-1 are pre- of the IBMX-treated cultures (Fig. 4E). Therefore, we conclude sented below. The growth rate of the ΔUmgpa3 mutant was sim- that UmGPA3 plays an important role in regulating pseudohy- ilar to that of the wild type in PDB (potato dextrose broth) phal growth in U. muhlenbergii, and the defect of the ΔUmgpa3 medium (SI Appendix,Fig.S9), suggesting that deletion of mutant in yeast-to-hypha transition can be rescued by treatment UmGPA3 gene had no effect on yeast growth. with IBMX. After incubation on 0.2× PDA for 10 d, the wild type formed colonies with melanized areas on which pseudohyphae or hyphae The UmGPA2 Gene Partially Regulates Pseudohyphal Growth. The protruded from the surface (Fig. 4A). Under the same condi- orthologs of UmGPA2 have overlapping functions with other Gα tions, the ΔUmgpa3 mutant formed colonies with a smooth subunits in Cryptococcus neoformans and Neurospora crassa (41, surface, likely due to its defects in the yeast-to-hypha transition. 42). To determine its function, we also generated the Umgpa2 In 10-d-old liquid BBM cultures, the majority of the wild-type deletion mutant by gene replacement. After incubation for 1 mo

Wang et al. PNAS Latest Articles | 5of12 Downloaded by guest on September 27, 2021 Fig. 3. Effects of cAMP or IBMX on colony morphology and cellular differentiation. (A) Colony and cell morphology of U. muhlenbergii after incubation on PDA with 10 mM cAMP for 14 d. Pseudohyphae were observed as early as 48 h and became longer after incubation for 72 h. In 14-d-old cultures, pseudohyphae became more abundant but generally not longer than eight compartments. (B) Cultures of U. muhlenbergii on PDA treated with IBMX were examined after incubation for 7 and 14 d. The filter paper placed on the left side of the U. muhlenbergii colony contained 25 nmol IBMX. Bottom shows a close-up view of the surface and margin of 14-d-old cultures. (C) Cellular differentiation induced by IBMX treatment was examined by DIC (differential interference contrast) microscopy. Individual cells were no longer visible in the heavily melanized and differentiated tissues at 14 dpt. (D) One-month-old PDA cultures 1 cm away from the IBMX filter were examined for surface differentiation, hyphal and pseudohyphal growth, and aggregates of melanized cells. SEM, scanning electron microscopy.

on 0.2× PDA, colony melanization and pseudohyphal growth observed in the wild type and ΔUmgpa2 mutant but never in the were still observed in ΔUmgpa2 cultures but to a lesser content in ΔUmgpa3 mutant (Fig. 5A). comparison with the wild type (Fig. 4A). In liquid BBM cultures, After cocultivation for 3 d, individual colonies consisting of pseudohyphal growth was still observed in the ΔUmgpa2 mutant, fungal and algal cells became visible. Whereas the vast majority but the length of its pseudohyphae was shorter than those of the (95.8%) of the wild-type U. muhlenbergii colonies-associated al- wild type (Fig. 4B). When cultured on PDA with 0.5 M sorbitol gal cells were in the pseudohyphal form, all of the ΔUmgpa3 for 10 d, the ΔUmgpa2 mutant also produced shorter pseudo- mutant colonies remained in the yeast form. Under the same hyphae and less wrinkled colonies than the wild type (Fig. 4 C conditions, 35.8% of ΔUmgpa2 mutant colonies underwent and D). These results indicate that UmGPA2 is not essential for morphological transition (Fig. 5B). After cocultivation for 10 d, the yeast-to-hypha transition but may play a minor role in the colonies with obvious pigmentation appeared on the wild-type growth of pseudohyphae. cultures, but ΔUmgpa3 mutant colonies remained nonpigmented and dome shaped (SI Appendix, Fig. S11). For the ΔUmgpa2 The ΔUmgpa3 Deletion Mutant Is Defective in Response to Algal Cells. mutant, the extent of changes in colony morphology and pig- To determine whether deletion of UmGPA3 affects its response mentation was between those of the wild type and the ΔUmgpa3 to the photobiont, yeast cells of the wild-type, ΔUmgpa2, and mutant strains (SI Appendix, Fig. S11). These results indicated ΔUmgpa3 mutant strains were cocultured with algal cells of T. that UmGPA3 plays a major role, but UmGPA2 also has a minor jamesii and observed every 12 h (Fig. 5). After 36 h of cocul- role in regulating the induction of pseudohyphal growth by algal turing, yeast cells of the wild type that were associated with algal cells of T. jamesii in U. muhlenbergii. cells began the yeast-to-hypha transition, resulting in the formation of pseudohyphae that became more distinctive by 72 h Dominant Active Mutations in UmGPA3 Promote Pseudohyphal (Fig. 5). Similar results were obtained with the ΔUmgpa2 mutant. Growth. The G142V and G355L mutations are known to result Under the same conditions, yeast cells of the ΔUmgpa3 mutant in dominant active mutations in Gpa2 of C. albicans (37). Se- closely associated with algal cells mainly grew by budding. Oc- quence alignment showed that these two amino acid residues are casionally, chains of three yeast cells were observed, but these highly conserved in UmGPA3 (G45 and G208) and its orthologs cells, unlike pseudohyphae, separated shortly thereafter. Pseu- from other ascomycetes (SI Appendix, Fig. S7). To further dohyphae with the tip cell growing away from algal cells were characterize its function in pseudohyphal growth, we generated

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Fig. 4. Defects of the ΔUmgpa3 mutant in pseudohyphal growth. (A) Ten-day-old 0.2× PDA cultures of the wild type and the ΔUmgpa2 and ΔUmgpa3 deletion mutants were examined for pigmentation and colonial growth. (B) Ten-day-old BBM cultures of the same set of strains were examined for pseudohyphal growth. Unlike the wild type and ΔUmgpa2 mutant, the ΔUmgpa3 mutant was defective in the yeast–pseudohypha transition. (C&D) The morphology of colonies (C) and cells (D) of the indicated strains cultured on PDA with 0.5 M sorbitol for 10 d. The wild type and ΔUmgpa2 formed wrinkly, fuzzy colonies and produced branching pseudohyphae. Pseudohyphal growth was not observed in smooth colonies of the ΔUmgpa3 mutant. (E) PDA cultures of the ΔUmgpa3 mutant treated with IBMX were examined for pseudohyphal growth and colony surface after incubation for 4 or 14 d.

the UmGPA3G45V and UmGPA3Q208L alleles and transformed These results indicated that the Q208L mutation may have a them into the wild type. The resulting UmGPA3G45V and more significant dominant active effect on UmGPA3 than the UmGPA3Q208L transformants grew normally as yeast cells on G45V mutation, which is similar to reports in C. albicans (37). PDA medium. The dominant active mutations in UmGPA3 had However, conidia were observed in all these transformants, in- no effect on growth (SI Appendix, Fig. S9). However, colony dicating that the G45V and Q208L mutations have no effect on melanization and pseudohyphal growth were observed in conidiation. 20-d-old PDA cultures of these two transformants. Whereas the wild type grew as yeast cells, the UmGPA3G45V and UmG- Dominant Active Mutations in UmGPA3 Affect the Establishment of PA3Q208L transformants, particularly the latter, had abundant the Initial Symbiotic Interaction. To test the effect of dominant pseudohyphae (Fig. 6A). These results indicated that expressing active mutations in UmGPA3 on symbiosis, the interaction be- these dominant UmGPA3 alleles enhanced pseudohyphal growth tween algal and yeast cells were monitored on PDA. In the wild- and may stimulate yeast-to-hypha transition under conditions not type strain, pseudohyphal growth was induced by algal cells of T. inducible in the wild type. jamesii. Green, healthy algal cells in close contact with pseudo- When cultured on PDA with 0.5 M sorbitol, colonies of the hyphae of U. muhlenbergii were observed at 72 h (Fig. 7A). In the UmGPA3G45V and UmGPA3Q208L transformants, particularly UmGPA3G45V and UmGPA3Q208L transformants, pseudohyphal the latter, had a bumpier surface and more hairy margins than growth also was observed, and the UmGPA3Q208L transformants wild-type colonies after incubation for 10 d (Fig. 6B). More ro- appeared to have more robust pseudohyphal growth. However, bust pseudohyphal growth was observed in the UmGPA3G45V after 72 h or longer, algal cells associated with pseudohyphae of and UmGPA3Q208L transformants than in the wild type (Fig. 6B). the UmGPA3G45V and UmGPA3Q208L transformants often were When cultured on BBM agar, long branching hyphae and collapsed and empty (Fig. 7A). Algal cells in close contact with pseudohyphae as well as aggregates of conidia were observed on U. muhlenbergii appeared to be damaged at the contact site by the margins of 20-d-old cultures of the UmGPA3G45V and the penetration or intrusion of fungal growth (Fig. 7A). In re- UmGPA3Q208L transformants (Fig. 6C). Under the same condi- peated assays, ∼30% of the algal cells associated with pseudo- tions, the wild type also produced conidia but had shorter hyphae of UmGPA3G45V and UmGPA3Q208L transformants were pseudohyphae (Fig. 6C). Clusters of pseudohyphae protruding not healthy or dead and lost chlorophyll autofluorescence by from the margins of UmGPA3Q208L colonies were longer and 72 h (Fig. 7 B and C). Under the same conditions, only 6.6% of more abundant than those in the UmGPA3G45V transformants. algal cells in cocultures with the wild-type U. muhlenbergii lost

Wang et al. PNAS Latest Articles | 7of12 Downloaded by guest on September 27, 2021 Fig. 5. Defects of the ΔUmgpa3 mutant in interactions with T. jamesii cells. (A) Time course assays of the interaction of the wild type and the ΔUmgpa2 and ΔUmgpa3 deletion mutants with the photobiont T. jamesii cells. Pseudohyphal growth was induced by T. jamesii in the wild type and ΔUmgpa2 mutant (marked with arrows). The ΔUmgpa3 mutant grew as yeast cells aggregated around algal cells but failed to form elongated pseudohyphae. (B) Percentages of colonies with extensive pseudohyphae in cocultures of T. jamesii cells with yeast cells of the wild type and ΔUmgpa2 or ΔUmgpa3 mutant after incubation for 3 d. Mean and SD were calculated with data from three independent replicates, with at least 100 cell clusters examined in each replicate. Asterisks * and** indicate significant differences based on one-way ANOVA with the LSD (least significant difference) t test analysis (P < 0.01).

chlorophyll autofluorescence (Fig. 7C), likely due to natural aging cultures or by growth on the inorganic medium BBM death of algal cells. These results indicate that these two domi- without a carbon source. Longer pseudohyphae bearing conidia nant active mutations in UmGPA3 may disrupt the initial es- were observed in BBM cultures, likely due to severe nutritional tablishment of their symbiotic relationship. deficiency. In Saccharomyces cerevisiae, nutrient limitation induces filamentation that is associated with the yeast to hyphal Discussion transition, although morphological changes are slightly different The yeast–hyphal dimorphism occurs to fungi belonging to dif- between the haploid and diploid cells (48, 49). U. muhlenbergii is ferent phyla and is usually stimulated by environmental factors a haploid fungus, and BBM medium lacks carbon and nitrogen (43). In fungal pathogens, including the human pathogen C. sources. Conidiation was not induced by cAMP or IBMX albicans and corn smut fungus U. maydis, the morphological treatment. Conidiation in axenic cultures has not been reported transformation occurs during infection and is necessary for ad- in U. muhlenbergii, but its lichen thalli produce hyaline unicel- aptation to host cells (44–47). Similar to a previous report (25), lular, rod-like spermatia that are formed in spermogonia (28). in this study, we isolated the U. muhlenbergii mycobiont that grew Because they are formed under different conditions and by dif- as yeast cells in culture from lichen thalli collected in China and ferent tissues, the relationship between conidia and spermatia is the United States, indicating that dimorphism is not an isolate- not clear. specific phenomenon but a common characteristic of U. muh- Like fungal pathogens that undergo dimorphic changes during lenbergii, a member of Lecanoromycetes, the largest class of infection, U. muhlenbergii also responded to its compatible lichen-forming fungi. photobiont with the dimorphic transition. Pseudohyphal growth Although U. muhlenbergii normally grew as unicellular yeast appeared to begin in yeast cells in close contact with T. jamesii cells, pseudohyphal growth was induced by nutrient limitation in cells as early as 36 h of cocultivation. As the time of cocultivation

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Fig. 6. Effects of dominant active mutations in UmGPA3 on pseudohyphal growth. (A) Twenty-day-old cultures of the wild type and transformants expressing the indicated dominant active alleles of UmGPA3 on PDA were examined for pseudohyphal growth. In comparison with the wild type that had limited pseudohyphal growth, the UmGPA3Q45V and UmGPA3Q208L transformants had more abundant and longer branching pseudohyphae, particularly the latter. (B) Ten-day-old cultures of the wild type and UmGPA3Q45V or UmGPA3Q208L transformant grown on PDA with 0.5 M sorbitol were examined for colony margins (Upper) and pseudohyphal growth (Lower). (C) Microscopic examination of colony edges of the indicated strains cultured on BBM plates for 20 d. Whereas the wild type had limited pseudohyphal growth, the UmGPA3Q45V and UmGPA3Q208L transformants, particularly the latter, produced long branching pseudohyphae. Aggregates of conidia (marked with arrows) were observed in all three strains.

increased, clusters of pseudohyphae tended to envelop algal two Gα subunits and found that the ΔUmgpa3 mutant was de- cells, and algal cells inside appeared to be compressed. Although fective in the dimorphic transition induced by stress or the it is not clear whether haustoria are formed, there was an in- presence of algal cells but still responded to IBMX for pseudo- terface and close association between mycobiont and photobiont hyphal growth. Furthermore, pseudohyphal growth was en- cells. We noticed that pseudohyphae and algal cells in close hanced in transformants expressing the UmGPA3G45V and contact could not be easily separated by dispersing in sterile UmGPA3Q208Ldominant active alleles. UmGpa3 is orthologous distilled water. Furthermore, pseudohyphae growing away from to MagB of M. oryzae and GzGpa3 of Fusarium graminearum, algal cells differed from those in close contact with algal cells in which are the major Gα subunits for regulating plant infection compartment length and morphology. In similar cocultivation processes (55–57). Its orthologs also are important for viru- assays with the Elliptochloris isolate, pseudohyphal growth was not lence in human pathogenic fungi and regulate the yeast-to- induced, and the mixture of U. muhlenbergii and Elliptochloris cells pseudohypha/hypha dimorphic transition in S. cerevisiae and C. could be easily separated by dispensing in water, indicating a albicans (37, 38, 41). In U. maydis, Gpa3 regulates both virulence specific recognition of photobiont cells by the mycobiont to induce and mating that are related to the dimorphic switch (39). Our pseudohyphal growth. In two dimorphic plant pathogenic fungi, U. results indicated that, like fungal pathogens, UmGpa3 functions maydis and Taphrina deformans, mating between compatible yeast upstream from the cAMP-PKA pathway for regulating pseudo- cells is associated with the formation of dikaryotic hyphae for hyphal growth and symbiosis in U. muhlenbergii. penetration and infectious growth (50, 51). In U. muhlenbergii,the In U. muhlenbergii, unlike the ΔUmgpa3 mutant, the ΔUmgpa2 yeast-to-pseudohypha transition may be necessary for the estab- deletion mutant still underwent dimorphic transition in response lishment of symbiosis and require the presence of compatible to nutrient or osmotic stress and algal cells. However, pseudo- photobiont cells. hyphal growth in the ΔUmgpa2 mutant was not as robust as in Similar to its role in the regulation of dimorphism in S. cer- the wild type. These results indicate that UmGPA2 may play a evisiae and several other fungi (52, 53), the well-conserved minor role in pseudohyphal growth. Therefore, it will be im- cAMP-PKA pathway was found to be important for regulating portant to generate the ΔUmgpa2 ΔUmgpa3 double mutants to pseudohyphal growth and possibly the establishment of the characterize the functional relationship between these two Gα symbiotic state in U. muhlenbergii. Because deletion of the reg- subunits during dimorphism and symbiosis. Unfortunately, ulatory or catalytic subunits of PKA often results in severe hygromycin resistance is the only selectable marker that is ef- growth defects in other fungi (54), in this study we characterized fective for transformant selection in this lichen-forming fungus,

Wang et al. PNAS Latest Articles | 9of12 Downloaded by guest on September 27, 2021 Fig. 7. Effects of dominant active mutations in UmGPA3 on interaction with algal cells. (A) Time course assays of the interaction of T. jamesii cells with the wild-type U. muhlenbergii and transformants expressing the indicated dominant active mutant alleles of UmGPA3. The same field was observed every 12 h. Dominant active mutations in UmGPA3 resulted in the collapse of algal cells after coincubation for 72 h. (B) Viability of T. jamesii cells decreased when cocultured with transformants expressing dominant active UmGPA3 alleles. After cocultivation for 3 d, algal cells in close associations with pseudohyphae were examined for cellular morphology and chlorophyll fluorescence (559-nm excitation, exposure time: 2,000 ms) by DIC (differential interference contrast) and epifluorescence microscopy. (C) Percentage of damaged algal cells with no or faint chlorophyll fluorescence in mycobiont–photobiont clusters in cocultures of T. jamesii cells with yeast cells of the wild type and Umgpa3G45V or Umgpa3G208L transformant after incubation for 3 d. Mean and SD were calculated with data from three independent replicates, with at least 100 algal cells examined in each replicate. *Significant differences between the wild type and Umgpa3G45V or Umgpa3G208L transformant based on one-way ANOVA with the LSD (least significant difference) t test analysis (P < 0.01).

although this is a report of generating targeted gene deletion graminearum, and other fungi to be dispensable for growth and mutants in U. muhlenbergii and lichenized fungi in general. The development (55–57). UmGPA1 gene was not characterized in this study because it is Although lichen thalli formed on the surface of rocks can tol- more distantly related to UmGPA3 than UmGPA2 (SI Appendix, erate desiccation and other stress, we found that U. muhlenbergii is Fig. S7), and its orthologs have been shown in M. oryzae, F. sensitive to hyperosmotic, oxidative, and cell wall stresses, which

10 of 12 | www.pnas.org/cgi/doi/10.1073/pnas.2005109117 Wang et al. Downloaded by guest on September 27, 2021 may be related to physiological differences between fungal cells responsible for algal cell death caused by the G45V and Q208L in axenic culture and lichen thalli. Nevertheless, moderate os- mutations in UmGPA3 in future studies. In some lichens, other motic stress such as 0.5 M sorbitol induced pseudohyphal growth. than wrapping around algal cells, the mycobiont may penetrate In fungi, responses to hyperosmotic stress are normally regulated and form small haustoria for nutrient uptake (15, 21). If U. by the high-osmolarity glycerol response (HOG) pathway (58, muhlenbergii penetrates and forms haustoria in T. jamesii cells, it 59). In U. muhlenbergii, the HOG pathway may cross-talk with will be important to determine whether these two dominant active the cAMP-PKA pathway for regulating the yeast-to-hypha mutations affect the proper regulation of haustorium formation Aspergillus fumigatus transition. In , the HOG and cAMP-PKA and cause the death of algal cells due to aggressive fungal growth. pathways coregulate the mobilization and utilization of storage This is a report on functional characterization of genes in carbohydrates for cell wall biosynthesis (60). In S. cerevisiae, lichen-forming or lichenized fungi. The approaches developed in filamentation occurs in HOG pathway mutants under osmotic stress (61). In U. muhlenbergii, activation of the HOG pathway by this study can be used to further characterize the intimate inter- osmotic stress may somehow activate the cAMP-PKA pathway action between the mycobiont and photobiont, particularly at the and induce pseudohyphal growth. Because the mating MAP ki- early stages. Because of its relatively fast growth rate in culture in nase pathway is known to be involved in dimorphism in S. cer- comparison with other lichenized fungi and amenability to mo- evisiae and C. albicans (62, 63), it may also cross-talk with the lecular manipulations, U. muhlenbergii is uniquely suitable for cAMP-PKA or HOG pathway in the yeast-to-pseudohypha tran- studying the regulation of dimorphism and symbiotic interactions sition in U. muhlenbergii. The genome sequence of U. muhlenbergii with photobiont cells. Furthermore, our data showed that the (34) has the conserved mating MAP (mitogen-activated protein) cAMP-PKA pathway plays an important role in regulating the kinase pathway, although its mating system and mating behavior yeast-to-hypha transition, and proper regulation of pseudohyphal have not been characterized. growth is critical for the establishment of symbiotic relationship in G45V In cocultivation with T. jamesii,theUmGPA3 and U. muhlenbergii, a species in Lecanoromycetes that contains Q208L UmGPA3 transformants switched rapidly to pseudohyphal majority of lichen-forming fungi. growth upon contact with algal cells. Interestingly, many algal cells associated with pseudohyphae of these transformants appeared to Materials and Methods be collapsed and dead after 72-h coculturing. The dominant active The materials and methods described in detail in SI Appendix include mutations in UmGPA3 likely resulted in defects in the proper methods for the isolation and identification of mycobiont and algal strains, regulation of pseudohyphal growth in U. muhlenbergii,andthe culture conditions for inducing dimorphic changes in U. muhlenbergii,co- death of algal cells may be directly caused by aggressive growth of cultivation assays with fungal and algal cells, and generation of the MICROBIOLOGY G45V Q208L the UmGPA3 and UmGPA3 transformants. It is also ΔUmgpa2 and ΔUmgpa3 deletion mutants and transformants expressing possible that algal cells of T. jamesii normally signal back to U. the UmGPA3G45V and UmGPA3G208L alleles. The origins of lichen samples, muhlenbergii to turn off UmGPA3-mediated cAMP signaling, sequences, and primers (SI Appendix, Table S1) used in this study also were which is affected by these dominant active mutations. In M. oryzae, presented in SI Appendix. the G42R mutation in MagB stimulates appressorium formation but significantly reduces its virulence on rice plants (64). In F. Data Availability. All study data are included in the article and SI Appendix. graminearum, the Q20L mutation in GzGPA2 also reduces its virulence on wheat heads (56). Similar to these fungal pathogens, ACKNOWLEDGMENTS. We thank Dr. Larry Dunkle for critical reading of this dominant active mutations in UmGPA3 may stimulate pseudo- manuscript. We also thank Mr. Jason Hollinger for providing lichen samples U. muhlenbergii collected in North Carolina and Dr. Robert Seiler at Purdue University for hyphal growth of but have a negative impact on assistance with scanning electron microscopy. This work was supported by the establishment of its symbiotic relationship with T. jamesii. China Scholar Council Grant 201804910321 (to Y.W.), a graduate student Therefore, it will be important to determine the cellular interac- fellowship grant from Purdue Research Foundation, and National Natural tion between U. muhlenbergii and T. jamesii and the mechanism Science Foundation of China Grant 31770022 (to X.W.).

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